Transfer of Cholesterol and Other Lipids From a Lipid Nanoemulsion to High-density Lipoprotein in Heart Transplant Patients

Background: Beyond the first year after a heart transplant (HT) procedure, patients often develop dyslipidemias, which may be implicated in the genesis of transplant coronary heart disease. High-density lipoprotein (HDL) has a several anti-atherogenic properties, but the status of HDL in HT patients is still controversial. Nonetheless, determination of HDL cholesterol concentration is not sufficient for evaluation of the overall HDL protective role. In this study, a fundamental functional property of HDL, the ability to simultaneously receive the major lipid classes, was tested in HT patients. Methods: Twenty HT patients and 20 healthy normolipidemic subjects paired for gender, age and body mass index were studied. Blood samples were collected after 12-hour fasting for determination of plasma lipids, glucose, paraxonase I (PON 1) activity, HDL diameter and transfer of labeled lipids from an artificial nanoemulsion to HDL. Results: Plasma triglycerides (159 +/- 63 vs 94 +/- 35 mg/dl) and glucose (104 +/- 20 vs 86 +/- 10 mg/dl) were greater in HT patients than in control subjects. HDL cholesterol was lower and HDL diameter was smaller in the HT group (HDL cholesterol: 44 +/- 11 vs 55 +/- 15 mg/dl; HDL diameter: 8.8 +/- 0.6 vs 9.0 +/- 1.2 nm). PON 1 activity did not differ (87 +/- 47 vs 75 +/- 37 nmol/min/ml). The transfer rates of free cholesterol and cholesteryl esters were diminished in HT patients (HT: 8.4 +/- 1.2% and 3.8 +/- 0.6%; controls: 9.7 +/- 1.9% and 4.7 +/- 1.2%, respectively). Conclusions: The transfer of free cholesterol and cholesteryl esters to HDL is diminished in HT patients; disturbance in the ability of HDL to receive lipids may affect the anti-atherogenic properties of the lipoprotein. J Heart Lung Transplant 2009;28:1075-80. Copyright (C) 2009 by the International Society for Heart and Lung Transplantation.

Metabolic syndrome features small, apolipoprotein A-I-poor, triglyceride-rich HDL3 particles with defective anti-apoptotic activity

The metabolic syndrome (MetS) phenotype is typically characterized by visceral obesity, insulin resistance, atherogenic dyslipidemia involving hypertriglyceridemia and subnormal levels of high density lipoprotein-cholesterol (HDL-C), oxidative stress and elevated cardiovascular risk. The potent antioxidative activity of small HDL3 is defective in MetS [Hansel B, et al. J Clin Endocrinol Metab 2004;89:4963-71]. We evaluated the functional capacity of small HDL3 particles from MetS subjects to protect endothelial cells from apoptosis induced by mildly oxidized low-density lipoprotein (oxLDL). MetS subjects presented an insulin-resistant obese phenotype, with hypertriglyceridemia, elevated apolipoprotein B and insulin levels, but subnormal HDL-C concentrations and chronic low grade inflammation (threefold elevation of C-reactive protein). When human microvascular endothelial cells (HMEC-1) were incubated with oxLDL (200 jig apolipoprotein B/ml) in the presence or absence of control HDL subfiractions (25 mu g protein/ml), small, dense HDL3b and 3c significantly inhibited cellular annexin V binding and intracellular generation of reactive oxygen species. The potent anti-apoptotic activity of small HDL3c particles was reduced (-35%; p < 0.05) in MetS subjects (n = 16) relative to normolipidemic controls (n = 7). The attenuated anti-apoptotic activity of HDL3c correlated with abdominal obesity, atherogenic dyslipidemia and systemic oxidative stress (p < 0.05), and was intimately associated with altered physicochemical properties of apolipoprotein A-I (apoA-I-poor HDL3c, involving core cholesteryl ester depletion and triglyceride enrichment. We conclude that in MetS, apoA-I-poor, small, dense HDL3c exert defective protection of endothelial cells from oxLDL-induced apoptosis, potentially reflecting functional anomalies intimately associated with abnormal neutral lipid core content. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Development of a synthetic 7-ketocholesterol- rich emulsion resembling chylomicrons

Lipid emulsions that mimic natural lipoproteins help to understand the metabolism and the constitutional organization of circulating lipids. Chylomicrons synthesised by enterocyte cells usually contain oxysterols such as 7-ketocholesterol (7-KC). Here we describe the development of a 7-KC-containing emulsion as a model for oxisterol-rich chylomicron. Different amounts of 7-KC were used. Emulsion characteristics as effective diameter, lipid saturation with radiolabeled lipids was evaluated. In conclusion, the production of a synthetic 7-KC-rich emulsion resembling hylomicrons was feasible, being a model for in vivo metabolism studies.

A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3(DGV) specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1-3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cav(DGV) to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains

Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed the effects of abolishing GPI biosynthesis on rafts, caveolae, and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts, indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin-1 and 2, were up-regulated in GPI-deficient cells, in contrast to flotillin-I and GM1, which were expressed at normal levels. Additionally, the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels, suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up-regulation.

Proline- and acidic amino acid-rich basic leucine zipper proteins modulate peroxisome proliferator-activated receptor alpha (PPAR alpha) activity.

In mammals, many aspects of metabolism are under circadian control. At least in part, this regulation is achieved by core-clock or clock-controlled transcription factors whose abundance and/or activity oscillate during the day. The clock-controlled proline- and acidic amino acid-rich domain basic leucine zipper proteins D-site-binding protein, thyrotroph embryonic factor, and hepatic leukemia factor have previously been shown to participate in the circadian control of xenobiotic detoxification in liver and other peripheral organs. Here we present genetic and biochemical evidence that the three proline- and acidic amino acid-rich basic leucine zipper proteins also play a key role in circadian lipid metabolism by influencing the rhythmic expression and activity of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). Our results suggest that, in liver, D-site-binding protein, hepatic leukemia factor, and thyrotroph embryonic factor contribute to the circadian transcription of genes specifying acyl-CoA thioesterases, leading to a cyclic release of fatty acids from thioesters. In turn, the fatty acids act as ligands for PPARα, and the activated PPARα receptor then stimulates the transcription of genes encoding proteins involved in the uptake and/or metabolism of lipids, cholesterol, and glucose metabolism.

A growth hormone-releasing peptide that binds scavenger receptor CD36 and ghrelin receptor up-regulates sterol transporters and cholesterol efflux in macrophages through a peroxisome proliferator-activated receptor gamma-dependent pathway.

Macrophages play a central role in the pathogenesis of atherosclerosis by accumulating cholesterol through increased uptake of oxidized low-density lipoproteins by scavenger receptor CD36, leading to foam cell formation. Here we demonstrate the ability of hexarelin, a GH-releasing peptide, to enhance the expression of ATP-binding cassette A1 and G1 transporters and cholesterol efflux in macrophages. These effects were associated with a transcriptional activation of nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma in response to binding of hexarelin to CD36 and GH secretagogue-receptor 1a, the receptor for ghrelin. The hormone binding domain was not required to mediate PPARgamma activation by hexarelin, and phosphorylation of PPARgamma was increased in THP-1 macrophages treated with hexarelin, suggesting that the response to hexarelin may involve PPARgamma activation function-1 activity. However, the activation of PPARgamma by hexarelin did not lead to an increase in CD36 expression, as opposed to liver X receptor (LXR)alpha, suggesting a differential regulation of PPARgamma-targeted genes in response to hexarelin. Chromatin immunoprecipitation assays showed that, in contrast to a PPARgamma agonist, the occupancy of the CD36 promoter by PPARgamma was not increased in THP-1 macrophages treated with hexarelin, whereas the LXRalpha promoter was strongly occupied by PPARgamma in the same conditions. Treatment of apolipoprotein E-null mice maintained on a lipid-rich diet with hexarelin resulted in a significant reduction in atherosclerotic lesions, concomitant with an enhanced expression of PPARgamma and LXRalpha target genes in peritoneal macrophages. The response was strongly impaired in PPARgamma(+/-) macrophages, indicating that PPARgamma was required to mediate the effect of hexarelin. These findings provide a novel mechanism by which the beneficial regulation of PPARgamma and cholesterol metabolism in macrophages could be regulated by CD36 and ghrelin receptor downstream effects.

Amphotericin B associated with triglyceride-rich nanoemulsion: stability studies and in vitro antifungal activity

Amphotericin B (AB) is the standard drug for invasive fungal infection therapy. It has a broad spectrum of activity and it is the best antifungal available against most yeasts and molds. Its therapeutic use, however, is limited by significant side effects, leading to a low therapeutic index when it is used as the traditional formulation (Fungizone®). Due to self-association, AB can form pores in cholesterol-containing membranes. We propose a triglyceride-rich nanoemulsion as a delivery system for AB in low levels of aggregation to reduce the toxicity against host cells.

Influences of alpha-tocopherol on cholesterol metabolism and fatty streak development in apolipoprotein E-deficient mice fed an atherogenic diet

Although the role of oxidized lipoproteins is well known in atherogenesis, the role of vitamin E supplementation is still controversial. There is also little information about cholesterol metabolism (hepatic concentration and fecal excretion) in the new models of atherosclerosis. In the present study, we evaluated the effect of moderate vitamin E supplementation on cholesterol metabolism and atherogenesis in apolipoprotein E (apo E)-deficient mice. Apo E-deficient mice were fed an atherogenic diet containing 40 or 400 mg/kg of alpha-tocopherol acetate for 6 weeks. Total cholesterol in serum and liver and 3-OH-alpha-sterols in feces, and fecal excretion of bile acids were determined and histological analyses of aortic lesion were performed. A vitamin E-rich diet did not affect body weight, food intake or serum cholesterol. Serum and hepatic concentrations of cholesterol as well as sterol concentration in feces were similar in both groups. However, when compared to controls, the alpha-tocopherol-treated mice showed a reduction of about 60% in the atherosclerotic lesions when both the sum of lesion areas and the average of the largest lesion area were considered. These results demonstrate that supplementation of moderate doses of alpha-tocopherol was able to slow atherogenesis in apo E-deficient mice and to reduce atherogenic lipoproteins without modifying the hepatic pool or fecal excretion of cholesterol and bile acids.

Cholesterol-dependent hemolytic activity of Passiflora quadrangularis leaves

Plants used in traditional medicine are rich sources of hemolysins and cytolysins, which are potential bactericidal and anticancer drugs. The present study demonstrates for the first time the presence of a hemolysin in the leaves of Passiflora quadrangularis L. This hemolysin is heat stable, resistant to trypsin treatment, has the capacity to froth, and acts very rapidly. The hemolysin activity is dose-dependent, with a slope greater than 1 in a double-logarithmic plot. Polyethylene glycols of high molecular weight were able to reduce the rate of hemolysis, while liposomes containing cholesterol completely inhibited it. In contrast, liposomes containing phosphatidylcholine were ineffective. The Passiflora hemolysin markedly increased the conductance of planar lipid bilayers containing cholesterol but was ineffective in cholesterol-free bilayers. Successive extraction of the crude hemolysin with n-hexane, chloroform, ethyl acetate, and n-butanol resulted in a 10-fold purification, with the hemolytic activity being recovered in the n-butanol fraction. The data suggest that membrane cholesterol is the primary target for this hemolysin and that several hemolysin molecules form a large transmembrane water pore. The properties of the Passiflora hemolysin, such as its frothing ability, positive color reaction with vanillin, selective extraction with n-butanol, HPLC profile, cholesterol-dependent membrane susceptibility, formation of a stable complex with cholesterol, and rapid erythrocyte lysis kinetics indicate that it is probably a saponin.

Comparison of new microemulsion prepared "Pt-in-Ceria" catalyst with conventional "Pt-on-Ceria" catalyst for water-gas shift reaction

New "Pt-in-CeO2" catalyst prepared by microemulsion method is shown to give higher activity for a water-gas shift reaction but with no formation of CH4, the side product from hydrogenation of carbon oxides using a hydrogen-rich reformate as compared to conventional "Pt-on-CeO2" catalysts. Detailed characterization by DRIFT analysis and temperature programmed reduction presented in this work clearly suggest the ceria coverage on Pt inhibits the metal from forming a strong CO adsorption.

Differential uptake of subfractions of triglyceride-rich lipoproteins by THP-1 macrophages

It is well known that raised plasma triglycerides (TG) are positively linked to the development of coronary heart disease. However, triglycerides circulate in a range of distinct lipoprotein subtractions and the relative atherogenicity of these subtractions is not clear. In this study, three fractions of triglyceride rich lipoprotein (TRL) were isolated from normolipidaemic males according to their differing Svedberg flotation (S-f) rates: chylomicron (CM, S-f > 400), very low-density lipoprotein (VLDL)-1 (S-f 60-400) and VLDL-2 (S-f 20-60). These fractions were incubated with THP-1 monocyte-derived macrophages for determination of cholesterol and TG accumulation, in the presence and absence of the lipoprotein lipase (LPL) inhibitor orlistat. Expression of LDL receptor related protein (LRP) and apolipoprotein B48 receptor (apoB48R) was also examined in both differentiating monocytes, and monocyte-derived macrophages, incubated with TRL. VLDL-I caused a significantly greater accumulation of TG within macrophages compared to VLDL-2. Binding studies also tended to show a greater preference for VLDL-1. No change in expression of LRP or apoB48R was observed in fully differentiated macrophages incubated with VLDL-1, VLDL-2 or CM, although a greater expression of LRP mRNA was observed in differentiating monocytes exposed to VLDL-1, compared to those incubated with CM or VLDL-2. TG loading in response to all three TRL fractions was blocked by orlistat, suggesting that it is likely that the major pathway for uptake of TG was hydrolysis by LPL. Calculations suggested that direct uptake of particles accounts for between 12 and 25% of total TAG uptake. In conclusion, THP monocyte-derived macrophages demonstrate a preference for VLDL-1, both through the LPL pathway and by direct uptake of whole particles. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Greater enrichment of triacylglycerol-rich lipoproteins with apolipoproteins E and C-III after meals rich in saturated fatty acids than after meals rich in unsaturated fatty acids

Background: Although there is considerable interest in the postprandial events involved in the absorption of dietary fats and the subsequent metabolism of diet-derived triacylglycerol-rich lipoproteins, little is known about the effects of meal fatty acids on the composition of these particles. Objective: We examined the effect of meal fatty acids on the lipid and apolipoprotein contents of triacylglycerol-rich lipoproteins. Design: Ten normolipidemic men received in random order a mixed meal containing 50 L, of a mixture of palm oil and cocoa butter [rich in saturated fatty acids (SFAs)], safflower oil [n-6 polyunsaturated fatty acids (PUFAs)]. or olive oil [monounsaturated fatty acids (MUFAs)] on 3 occasions. Fasting and postprandial apolipoproteins B-48. B-100, E. C-II, and C-III and lipids (triacylglycerol and cholesterol) were measured in plasma fractions with Svedberg flotation rates (S-f) >400 S-f 60-400, and S-f 20 - 60. Results: Calculation of the composition of the triacylglycerol-rich lipoproteins (expressed per mole of apolipoprotein B) showed notable differences in the lipid and apolipoprotein contents of the SFA-enriched particles in the S-f > 400 and S-f 60-400 fractions. After the SFA meal, triacylglycerol-rich lipoproteins in these fractions showed significantly greater amounts of triacylglycerol and of apolipoproteins C-II (Sf 60-400 fraction only), C-III, and E than were found after the MUFA meal (P < 0.02) and more cholesterol, apolipoprotein C-III (Sf > 400 fraction only), and apolipoprotein E than after the PUFA meal (P < 0.02). Conclusions: Differences in the composition of S-f > 400 and S-f 60-400 triacylglycerol-rich lipoproteins formed after saturated compared with unsaturated fatty acid-rich meals may explain differences in the metabolic handling of dietary fats.

Impact of saturated, polyunsaturated and monounsaturated fatty acid-rich micelles on lipoprotein synthesis and secretion in Caco-2 cells

Meal fatty acids have been shown to modulate the size and composition of triacylglycerol (TAG)-rich lipoproteins influencing the magnitude and duration of the postprandial plasma TAG response. As a result there is considerable interest in the origin of these meal fatty-acid induced differences in particle composition. Caco-2 cells were incubated over 4 days with fatty acid mixtures resembling the composition of saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acid (PUFA)-rich meals fed in a previous postprandial study to determine their impact on lipoprotein synthesis and secretion. The MUFA- and PUFA-rich mixtures supported greater intracellular TAG, but not cholesterol accumulation compared with the SFA-rich mixture (P < 0.001). The MUFA-rich mixture promoted significantly greater TAG and cholesterol secretion than the other mixtures and significantly more apolipoprotein B-100 secretion than the PUFA-rich mixture (P < 0.05). Electron microscopy revealed the SFA-rich mixture had led to unfavourable effects on cellular morphology, compared with the unsaturated fatty acid-rich mixtures. Our findings suggest the MUFA-rich mixture, may support the formation of a greater number of TAG-rich lipoproteins, which is consistent with indirect observations from our human study. Our electron micrographs are suggestive that some endocytotic uptake of MUFA-rich taurocholate micelles may promote greater lipoprotein synthesis and secretion in Caco-2 cells.

Contribution of apolipoprotein E genotype and docosahexaenoic acid to the LDL-cholesterol response to fish oil

Objectives: To investigate the impact of apolipoprotein E (apoE) genotype on the response of the plasma lipoprotein profile to eicosapentaenoic acid (EPA) versus docosahexaenoic acid (DHA) intervention in humans. Methods and results: 38 healthy normolipidaemic males, prospectively recruited on the basis of apoE genotype (n = 20 E3/E3 and n = 18 E3/E4), completed a double-blind placebo-controlled cross-over trial, consisting of 3 × 4 week intervention arms of either control oil, EPA-rich oil (ERO, 3.3 g EPA/day) or DHA-rich oil (DRO, 3.7 g DHA/day) in random order, separated by 10 week wash-out periods. A significant genotype-independent 28% and 19% reduction in plasma triglycerides in response to ERO and DRO was observed. For total cholesterol (TC), no significant treatment effects were evident; however a significant genotype by treatment interaction emerged (P = 0.045), with a differential response to ERO and DRO in E4 carriers. Although the genotype × treatment interaction for LDL-cholesterol (P = 0.089) did not reach significance, within DRO treatment analysis indicated a 10% increase in LDL (P = 0.029) in E4 carriers with a non-significant 4% reduction in E3/E3 individuals. A genotype-independent increase in LDL mass was observed following DRO intervention (P = 0.018). Competitive uptake studies in HepG2 cells using plasma very low density lipoproteins (VLDL) from the human trial, indicated that following DRO treatment, VLDL2 fractions obtained from E3/E4 individuals resulted in a significant 32% (P = 0.002) reduction in LDL uptake relative to the control. Conclusions: High dose DHA supplementation is associated with increases in total cholesterol in E4 carriers, which appears to be due to an increase in LDL-C and may in part negate the cardioprotective action of DHA in this population subgroup.